Reliability assessment of methylthioadenosine phosphorylase immunohistochemistry as a surrogate biomarker for CDKN2A homozygous deletion in adult-type IDH-mutant diffuse gliomas

Abstract According to the 2021 World Health Organization classification of brain tumors, astrocytomas containing a CDKN2A/B homozygous deletion (HD) are designated as grade 4 even when no microvascular proliferation and/or necrosis is present. In this study, we aimed to investigate the relationship between CDKN2A HD and loss of methylthioadenosine phosphorylase (MTAP) expression in adult-type IDH-mutant gliomas and to assess the sensitivity and specificity of MTAP immunohistochemistry (IHC) along with interobserver agreement as a surrogate biomarker for CDKN2A HD. Eighty-eight astrocytomas and 71 oligodendrogliomas cases that were diagnosed between 2014 and 2021 at Hacettepe University were selected and tissue microarrays were conducted to perform CDKN2A fluorescence in situ hybridization and MTAP IHC. Twenty-five (15.7%) cases harbored CDKN2A HD. MTAP loss was detected in 28 (15.7%) cases by the first observer and 27 (17%) cases by the second observer. The sensitivity and specificity of MTAP were calculated as 88% and 95.52%-96.27% for 2 observers. A very good/perfect agreement was noted between the observers (Cohen kappa coefficient = 0.938). Intratumoral heterogeneity was observed in 4 cases. MTAP IHC was found to be a reliable surrogate biomarker as a possible alternative to CDKN2A HD identification with a high sensitivity and specificity along with high interobserver agreement.


I NTR ODUCT ION
Diffuse gliomas comprise a significant proportion of primary malignant intracranial tumors.In the 2021 World Health Organization (WHO) classification of central nervous system (CNS) tumors, diffuse glial tumors are categorized under adult and pediatric types (1).In the process that started with the discovery of isocitrate dehydrogenase (IDH) 1/2 (IDH1/2) mutations in 2009 (2), adult-type diffuse gliomas are divided into different subtypes based on IDH1/2 mutation and 1p/19q codeletion (3) status.According to the 2021 WHO, they are classified into 3 groups; IDH-mutant astrocytoma, IDH-mutant and 1p/19q codeleted oligodendroglioma, and IDH-wild type glioblastoma.
Recently, homozygous deletion (HD) of cyclin-dependent kinase inhibitors 2A and 2B (CDKN2A/B) genes has emerged as a biomarker that can be used to predict poor prognosis in diffuse astrocytic tumors.Many studies have revealed that CDKN2A/B HD is associated with adverse prognosis and reduced survival in IDH-mutant diffuse astrocytic gliomas (4)(5)(6).In the "cIMPACT-NOW" published in 2020, it was recommended that astrocytomas containing CDKN2A/B HD be considered WHO grade IV (2016) astrocytomas even if there is no microvascular proliferation (MVP) or necrosis (7).Based on these findings, according to the change in the grading of IDH-mutant astrocytomas in the 2021 WHO CNS tumors classification, tumors containing CDKN2A/ CDKN2B HD are classified as grade 4 even if they do not contain microvascular proliferation and/or necrosis (1).While CDKN2A/ B HD can be seen in some (<10%) of WHO CNS grade 3 oligodendrogliomas, it is not expected in grade 2 oligodendrogliomas, and its presence has been related to short survival times independent of MVP and necrosis (8).Therefore, CDKN2A/B HD can be used as a molecular aid in oligodendroglial tumors that are difficult to grade.
CDKN2A and CDKN2B genes are adjacent to each other on chromosome 9p21.The CDKN2A gene plays a role in encoding p16 and p14 tumor suppressor proteins, while the CDKN2B gene encodes p15 protein.In this way, they inhibit the transition of cells from the G1 to the S phase and MDM2 activity that causes p53 degradation (9,10).CDKN2A/B pathway alterations contribute to the development of various malignant tumors.Currently, CDKN2A HD can usually be evaluated using molecular methods such as fluorescence in situ hybridization (FISH), comparative genomic hybridization, multiplex ligation-dependent probe amplification, nextgeneration sequencing, or DNA methylation-based profiling.However, because of the high cost, long reporting period, and difficulty in accessing these sophisticated molecular methods, more accessible and low-cost methods such as immunohistochemical stains are needed.
There are controversial results on the reliability of p16, the loss of which has long been thought to be a possible surrogate marker for CDKN2A in glial tumors (11)(12)(13).Recently, there have been studies conducted with mesothelioma, various epithelial tumors, meningioma, and pleomorphic xanthoastrocytoma (PXA) in order to use methylthioadenosine phosphorylase (MTAP) immunohistochemical expression loss as a surrogate biomarker to detect CDKN2A HD status (14-21).The MTAP gene, which is located on chromosome 9p.21 and 165 kb telomeric to the CDKN2A gene, shows HD along with CDKN2A HD in 80%-90% of cases.The MTAP protein is present in every cell and has a crucial role in adenosine monophosphate and methionine synthesis.When the MTAP gene is HD, normally expected cytoplasmic MTAP protein expression is lost and this can be detected by IHC (22,23).The anticipated role of MTAP IHC in diffuse gliomas, as an alternative to CDKN2A HD, has not yet been fully explored; there are only 2 published studies in the literature.In 2020, Satomi et al reported 88% sensitivity and 98% specificity for MTAP IHC in predicting CDKN2A HD in IDH-mutant astrocytomas.They also found that loss of MTAP expression was associated with adverse prognosis and shorter survival times similar to CDKN2A HD (24).Three years later, Maragkou et al (25) reported that cytoplasmic MTAP expression loss had 100% sensitivity and 97% specificity in their glioma cohort.
With the 2021 WHO classification of brain tumors, the frequent use of expensive methods has become inevitable but since it will not be possible to examine the genomic and proteomic features of all brain tumors, especially in low-and middle-income countries, an approach in which immune surrogate biomarkers can be used would be beneficial.
Our aim in this study was to assess the relationship between CDKN2A HD and loss of MTAP expression in adult-type IDH-mutant diffuse gliomas and determine the sensitivity and specificity of MTAP immunohistochemistry (IHC) as a surrogate biomarker for FISH assay detection of CDKN2A deletion of 2 independent observers.To the best of our knowledge, this would be the first study to evaluate interobserver agreement and intratumoral heterogeneity of MTAP staining in gliomas, along with its sensitivity and specificity.

MAT E RI ALS AND ME T HODS Patient selection
This

Tissue microarray
To prepare tissue microarrays (TMAs), representative tumor paraffin blocks belonging to each case were selected.Two 3mm-diameter cores of tumor tissue were taken out from the selected blocks with the help of a manual punch arrayer and the cores were placed in new receiver blocks.Eleven TMA blocks were constructed, and 4-lm multiple sections were taken from each.One of them was stained H&E to check the presence of tumoral tissue under a light microscope.

Immunohistochemistry
TMA sections were stained with MTAP antibody (Abcam, Cambridge, United Kingdom, clone EPR6893, Targeted Retrieval Solution, pH 9.0, 1:1000 dilution, 25 minutes incubation) using Leica Bond-Max (Leica Microsystems, Wetzlar, Germany) automated IHC platform, according to the manufacturer's instructions.As an external control, colon tissues were used in every block.Cytoplasmic staining with or without nuclear staining was accepted as preserved MTAP expression (Fig. 1A-C).Complete loss of cytoplasmic staining with adequate positive internal controls (endothelial, inflammatory, and non-neoplastic glial cells) was accepted as loss of MTAP expression (Fig. 1D).MTAP staining was evaluated and scored by 2 pathologists (F.S. and F.G.) independently for both tumor cores of each case.

Fluorescence in situ hybridization
For CDKN2A FISH analysis, Zytolight SPEC CDKN2A/CEN 9 Dual Color Probe was used on TMA sections according to the manufacturer's instructions.CDKN2A FISH analysis was performed on both tumor cores of each case.The prepared slides were examined under a fluorescence microscope.The CDKN2A probe (test probe) was expected to give green, and the CEN9 probe (reference probe) would give orange signal.Two orange and 2 green signals were expected in a normal interphase nucleus.One hundred non-overlapping cells were counted in a dark field.
CDKN2A HD was defined as the case that the number of cells containing at least one orange signal and missing both green signals was greater than 10%.CDKN2A heterozygous deletion was defined as a test probe/reference probe ratio 0.8 and the percentage of cells with a reference probe/test probe ratio of 2 was !35%.CDKN2A amplification was defined as a test probe/reference probe ratio !2.

Statistical analysis
Statistical analyses were performed in SPSS version 25.0 (IBM, Armonk, NY), and p < 0.05 was considered statistically significant.The normal distribution of continuous data was evaluated by visualization of histograms and the Shapiro-Wilk test.Normally distributed continuous data were reported as mean, standard deviation, and categorical data as numbers and percentages.The difference between 2 independent groups of continuous data was evaluated with the independent t-test.Chi-square or Fisher exact test investigated the relationship between categorical variables.Sensitivity, specificity, positive and negative odds ratio, positive and negative predictive ratio, and precision were evaluated with 95% confidence intervals to assess the diagnostic accuracy of the tests.The agreement between the 2 observers was reviewed by the Kappa coefficient and interpreted as follows: <0.2, slight; 0.2 to <0.4, fair; 0.4 to <0.6, moderate; 0.6 to <0.8, substantial/good; 0.8 to <1.0, very good/perfect.

RES U LT S Clinical and histopathological features
Clinicopathological findings are summarized in Table 1.In this study, 159 adult-type IDH-mutant diffuse glioma cases belonging to 147 patients were evaluated.Fifty-two (35.3%) of 147 patients were female, and 95 (64.6%) were male.The mean age for astrocytoma was 44.3 and 34.9 years for oligodendroglioma.

FISH and IHC results
The results of CDKN2A FISH and MTAP IHC are summarized in Table 2.The CDKN2A status of 159 tumors was evaluated by FISH.CDKN2A HD was detected in 25 (15.7%) of 159 tumors.In astrocytomas, 20 (22.7%) of 88 cases had CDKN2A HD, while 5 (7%) of 71 oligodendrogliomas showed this deletion.The 2016 WHO CNS grades of the astrocytomas that harbor CDKN2A HD were grade II in 2 tumors, grade III in 8 tumors, and grade IV in 10 tumors.After evaluating CDKN2A HD status, the WHO CNS grade of 10 tumors was changed to WHO CNS grade 4 according to the WHO CNS 2021 criteria.In the oligodendrogliomas, none of the WHO CNS grade 2 tumors had CDKN2A HD, while 5 (11.6%) of 43 grade 3 cases showed CDKN2A HD.
Regarding MTAP expression, 159 tumors were scored by 2 observers.The first observer (F.S.) assessed 28 (15.7%) of them, and the second observer (F.G.) assessed 27 (17%) of them as having a loss of MTAP expression.In the astrocytoma group, the first observer detected MTAP loss in 23 (23.9%) of the tumors, and the second observer detected MTAP loss in 22 (23.9%) of them.In the oligodendroglioma group, MTAP loss was detected in 7 (9.9%) of 73 tumors evaluated.There were only 3 cases that did not show interobserver agreement due to heterogenous staining patterns characterized by the intermingling of stained and non-stained cells and in which identifying which cells were neoplastic and which were not was not entirely clear on first evaluation.However, after the re-evaluation of these MTAP stainings with their H&E counterparts, it was decided that this heterogenous staining reflected intratumoral heterogeneity.
CDKN2A heterozygous deletion was found in 3 of 88 astrocytomas (3.4%) while 3 cases (3.4%) showed CDKN2A amplification.In none of these cases, MTAP expression loss was observed.No CDKN2A heterozygous deletion or amplification was detected in oligodendrogliomas.
When the interobserver agreement for the detection of MTAP loss was evaluated with Cohen kappa coefficient, a very good/perfect agreement of 0.938 was found between the 2 observers.

Correlation between CDKN2A HD and loss of MTAP expression
The comparison of CDKN2A HD and MTAP expression is shown in Table 3.When the relationship between the presence of CDKN2A HD and loss of MTAP expression in both observers was evaluated with the Fisher exact test, it was found that MTAP loss was higher in the presence of CDKN2A HD (p < 0.001).
When the sensitivity and specificity of MTAP immunohistochemical staining loss were calculated as an alternative to the CDKN2A FISH in all cases, the sensitivity for the first observer was 88.00%, and the specificity was 95.52%.In comparison, the sensitivity for the second observer was 88.00%, and the specificity was 96.27%.Among the astrocytomas, the sensitivity of MTAP for the first observer was 90%, and the specificity was 95.59%, while the sensitivity for the second observer was 85%, and the specificity was 95.59%.In the oligodendrogliomas, the sensitivity of MTAP for the first observer was 80%, and the specificity was 95.45%, while the sensitivity for the second observer was 100%, and the specificity was 96.97%.
There were also cases showing discordance between CDKN2A status and MTAP expression.MTAP loss was observed in 22 of 25 tumors that have CDKN2A HD; but not detected in 3 of them (Fig. 2A, B).Meanwhile, MTAP expression loss was noted in 5 of 134 tumors without CDKN2A HD (Fig. 2C, D).When the relationship between CDKN2A HD or MTAP loss and patient age, gender, and tumor location was analyzed, no correlation was found.

DI SC USSI ON
Adult-type IDH-mutant gliomas are divided into 2 major groups on the basis of 1p/19q status: IDH-mutant astrocytoma, and IDH-mutant and 1p/19q codeleted oligodendroglioma.According to the recent change of IDH-mutant astrocytoma grading in the 2021 WHO Classification of CNS tumors, tumors containing CDKN2A/B HD are accepted as grade 4 even if they do not contain MVP and/or necrosis (1).Therefore, assessment of CDKN2A/B HD status is mandatory for accurate grading of diffuse astrocytic tumors lacking necrosis and/or MVP.In oligodendrogliomas, CDKN2A/B HD, which is not seen in WHO CNS grade 2 tumors but can be seen in some grade 3 tumors, has been found to be related to short survival times independent of MVP and necrosis (8).
Overall, we detected CDKN2A HD in 25 (15.7%) of 159 tumors.This rate was similar to the "French national POLA network" cohort (13.9%, 127/911) reported by Appay et al (8).CDKN2A HD was detected in 20 (22.7%) of 88 astrocytoma cases.Although this rate was higher than the rates of the cohorts of Appay et al (11%, 47/428) and Shirahata et al (5) (14%, 31/224); it was lower than the cohort of Korshunov et al (26) (43%, 42/97).Due to the presence of CDKN2A HD, the CNS WHO grade of 10 (11.3%)IDH-mutant astrocytomas was upgraded to grade 4 (2 were previously grade II, 8 were previously grade III according to the WHO 2016 at the time of diagnosis).In oligodendrogliomas, CDKN2A HD was detected in 7.04% (5/71) of tumors; all were WHO CNS grade 3 tumors.CDKN2A HD was not detected in any of the grade 2 tumors.In 2020, Appay et al (8) reported that CDKN2A HD was seen in 7% (33/483) of grade 3 oligodendrogliomas, similar to our result, and CDKN2A HD in oligodendroglial tumors was associated with shorter overall survival independent of necrosis and MVP.Therefore, based on these results, it might be useful to determine CDKN2A HD status in oligodendroglial tumors with difficulty in grading.
There are suggestions in the literature that MTAP immunohistochemical staining can be utilized as a surrogate biomarker for molecular methods in detecting CDKN2A HD (14-21).However, there are a limited number of studies conducted on diffuse gliomas (24,25).In our study, the sensitivity of MTAP was 88% for both observers, and the specificity was 95.52%-96.27%,respectively.This finding is consistent with the 2 other studies in diffuse gliomas reported by Satomi et al (24) (88% sensitivity and 98% specificity), and Maragkou et al (25) (97% sensitivity and 100% specificity).Similarly, in a recent study with 23 PXA cases, the sensitivity of MTAP IHC was found to be 86.7% and the specificity 100% (18).In a study of 30 meningioma cases published by Sasaki et al (20), MTAP loss was found in all 5 cases in which CDKN2A HD was detected, and they reported sensitivity and specificity of MTAP as 100%.With these findings, we can conclude that MTAP IHC is a reliable surrogate biomarker that shows high sensitivity and specificity for detecting CDKN2A HD in diffuse gliomas as well as other CNS tumors.
However, some cases show a discrepancy between CDKN2A status and MTAP expression.In our study, loss of MTAP staining was observed in 22 of 25 cases that have CDKN2A HD; in 3 of them, MTAP loss was not detected.In 80%-90% of cases, deletion in chromosome 9p21 is associated with HD of CDKN2A and HD of MTAP gene, which is 165 kb telomeric to CDKN2A (22).However, depending on the extent of the deletion, sometimes CDKN2A HD may not be accompanied by MTAP HD.According to a study including 230 glioma cases, among 9p21 deleted cases 10% had CDKN2A HD without MTAP HD (23).In a study with pancreatic and periampullary cancers, in which CDKN2A HD is common (40%), it was reported that CDKN2A HD was seen in half of the cases without MTAP HD (16).Therefore, in some cases, there is no immunohistochemical loss of MTAP as MTAP HD does not accompany existing CDKN2A HD.Based on these findings, this situation might be valid for the 3 cases in our series that did not show MTAP loss despite CDKN2A HD.The conclusion to be drawn here is that retained MTAP expression does not always rule out CDKN2A HD.
In our series, MTAP expression loss was observed in 5 of 134 cases without CDKN2A HD.In the literature, rare cases (0.5%) of MTAP HD without CDKN2A HD have been reported (23).This situation might be explained either by MTAP HD without CDKN2A HD or another MTAP protein inactivation mechanism rather than HD (27).In conclusion, loss of MTAP staining does not always indicate the presence of CDKN2A HD.Interobserver agreement of MTAP expression loss between 2 independent observers was calculated by Kappa coefficient analysis in our study; an almost perfect agreement of 0.938 was found.Similarly, Chapel et al (14) showed very good and good agreement, respectively, between 4 observers (Kappa coefficient 0.85) and 2 laboratories (Kappa coefficient 0.77) in MTAP staining in mesothelioma cases.Therefore, MTAP IHC emerges as a reliable surrogate biomarker with high interobserver agreement.
The 2 most used clones for MTAP IHC are "EPR6893" and "2G4."In this study, clone EPR6893 was used, and immunohistochemical staining was able to be evaluated optimally in neoplastic cells and internal controls in most of the existing cases.Similarly, Lou et al (18) reported that they obtained effective staining with the "EPR6893" clone in PXA cases.However, Satomi et al reported that they could easily evaluate 61% of the cases with the "EPR6893" clone and 64% with the "2G4" clone in their diffuse glioma series.They also mentioned that the staining was stronger with the "2G4" clone even though they encountered similar difficulties for both clones when scoring the staining (24).The major problem in assessing the MTAP IHC is interpreting the absence of staining.Insufficient tissue fixation and technical issues during the staining procedure may lead absence of MTAP staining which can be interpreted as a false positive result.It is crucial to ensure that internal controls such as endothelial, inflammatory, and non-neoplastic glial cells are stained before evaluating MTAP loss.It will be wise to perform CDKN2A FISH in the suspicious cases.
To observe intratumoral heterogeneity in MTAP IHC and CDKN2A FISH, TMA blocks were prepared by taking 2 separate cores from different areas for each tumor.In the evaluation of MTAP IHC, intratumoral heterogeneity was observed in 4 cases, while MTAP immunohistochemical staining was homogeneous for both cores in the remaining cases.Three of 4 cases showed intermingling stained and non-stained cells.Only in one case, a sharp demarcation of loss of MTAP expression supported by CDKN2A FISH heterogeneity was observed.As illustrated in Figure 3, CDKN2A HD was absent in tumor areas where MTAP expression was preserved and CDKN2A HD was observed in tumor cells with MTAP loss.The remaining 158 cases did not show intratumoral heterogeneity in the CDKN2A FISH examination.In contrast to our results, Maragkou et al (25) reported that they did not observe any heterogeneity in MTAP staining in their glioma cohort.However, some studies using DNA methylation techniques have reported that intratumoral heterogeneity can be seen in diffuse gliomas (28,29).Among the intratumoral heterogeneity for CDKN2A/B HD, Vega et al (28) mentioned in their study that they evaluated intratumoral heterogeneity with the help of DNA methylation profile in 39 cases of glioma and meningioma, and they reported that they detected intratumoral heterogeneity in CDKN2A/B HD in 1 of the 2 IDH-mutant astrocytomas included in the study, and this may lead to incorrect grading of IDH-mutant astrocytomas.So, the intratumoral heterogeneity seen in MTAP staining in some cases may reflect the heterogeneity seen in CDKN2A HD.Therefore, the possibility of intratumoral heterogeneity should be kept in mind when evaluating MTAP IHC, as well as the assessment of CDKN2A HD by FISH.
In addition to accompanying CDKN2A HD, there are also publications indicating that MTAP inactivation contributes to gliomagenesis through epigenetic mechanisms, especially in glioblastomas (30); thus, MTAP may be a target for treatment (30,31).In the future, with the elucidation of the underlying mechanisms and the development of new targeted therapies, MTAP might be used as a marker that has the potential to be encountered with its therapeutic features as well as its diagnostic and prognostic features.
In summary, MTAP immunohistochemical staining seems to be a reliable surrogate biomarker that has high sensitivity and specificity for detecting CDKN2A HD and shows high interobserver agreement.It is essential to be aware of intratumoral heterogeneity while evaluating both CDKN2A HD and MTAP loss in order to make a correct assessment.

ET H IC AL APP RO VAL
This study was conducted with appropriate ethics committee approval: Hacettepe University Non-interventional Research Ethics Committee; 2021/17-08.

Figure 1 .
Figure 1.Evaluation of MTAP staining.(A) Strong cytoplasmic expression.(B) weak cytoplasmic expression.(C) Nuclear staining along with cytoplasmic staining.(D) Cytoplasmic expression loss with positive internal control staining.All are at 200Â magnification.

Figure 3 .
Figure 3. Demonstration of intratumoral heterogeneity.(A) There is a sharp demarcation between lost and retained expression of MTAP staining.(B) CDKN2A HD is not seen in tumor cells with retained MTAP expression.(C) There is CDKN2A HD in tumor cells with MTAP loss.In the FISH images, green signals represent CDKN2A; orange signals represent CEN9.

Table 2 .
CDKN2A FISH And MTAP IHC results

Table 3 .
Comparison of CDKN2A HD and MTAP expression